Antibiotic 5057B substance

ABSTRACT

New antibiotic 5057B and process for its preparation. Antibiotic 5057B itself is an acidic material, and its sodium salt has the following physical and chemical properties: 
     (1) Colorless needles 
     (2) Melting point: 143°-145° C. 
     (3) Elementary analysis (Found %): C, 59.96; H, 9.03; O, 27.60; Na, 3.41 
     (4) Specific rotation: [α] D   25  =+5.5° (C 1, CHCl 3 ) 
     (5) Maximum absorption in ultraviolet absorption spectrum: λ max   MeOH  (E 1  cm 1% ) 290 nm (0.88) 
     (6) Characteristic absorption (cm -1 ) in infrared absorption spectrum (taken with the potassium bromide tablet: 3480, 3000, 2970, 2812, 1719, 1645, 1595, 1465, 1385, 1160, 1100, 1035, 980 
     (7) Solubility: soluble in methanol, ethanol, ethyl acetate, chloroform, ether, acetone, and benzene and so forth; insoluble in water 
     (8) Color reaction: positive in the 2,4-dinitrophenylhydrazine reaction; negative in the ninhydrin reaction and the vanilline-sulfuric acid reaction; colors with I 2  gas. 
     The antibiotic is prepared by culturing Streptomyces bacteria, and collecting the antibiotic from the culture product.

FIELD OF THE INVENTION

The present invention relates to a new antibiotic 5057B substance and aprocess for production thereof.

DESCRIPTION OF THE PRIOR ART

The present inventors previously found that an actinomycete Streptomycessp. 5057 strain belonging to the genus Streptomyces (FERM BP-62)produces a substance having an antibacterial activity in its culturebroth and isolated an antibiotic 5057(A) from the culture broth (referto the specification of Japanese Patent Publication No. 8641/1979).

The present inventors, in the course of further researches, have foundthat a new substance different from the 5057A substance is present insaid culture broth and isolated the same, which was designated as the5057B substance.

The 5057B substance, in view of its physicochemical and biologicalproperties, is an antibiotic belonging to the antibiotic group ofpolyether series. Among antibiotics of polyether series, there areheretofore known, as those showing ultraviolet absorption in thevicinity of 290 nm, lysocellin (The Journal of Antibiotics, Vol. 28,Pages 118-121, 1975) and the 5057A substance (Japanese PatentPublication No. 8641/1979); however, the 5057B substance is a newantibiotic which differs from the above-mentioned known antibiotics inthe melting point, specific rotation, infrared absorption spectrum, andthe Rf value on silica gel thin layer chromatography.

DISCLOSURE OF THE INVENTION

The present invention relates to a new antibiotic 5057B substance havinga potent antibacterial activity, especially against gram-positivebacteria, and a process for production thereof comprising cultivating amicrooragnism, belonging to the genus Streptomyces, which has theability to produce the 5057B substance and then recovering the 5057Bsubstance from the culture broth.

The strain Streptomyces sp. 5057, belonging to the genus Streptomyces,employed in the production of the 5057B substance was deposited on May1, 1981 with Fermentation Research Institute, Agency of IndustrialScience and Technology (No. 1-3, Yatabechohigashi 1-chome, TsukubaDistrict, Ibaragi Prefecture, Japan) and accorded the accession numberFERM BP-62. Microbiological properties of the strain is described indetail in the specification of Japanese Patent Publication No.8641/1979.

In order to obtain the 5057B substance according to the presentinvention, ordinary methods for cultivation of actinomycetes can beemployed, but a cultivation with aeration-agitation is suited for theindustrial production. The cultivation temperature of 25°-35° C. isusual, but the temperature of 30° C. is preferable. As a culture medium,there can be used one customarily employed for cultivation ofmicrooragnisms belonging to the genus Streptomyces, for example, onecontaining carbon sources such as glucose, starch, glycerol, dextrin,sucrose, and animal or vegetable oils and nitrogen sources such assoybean meal, corn steep liquor, wheat embryo, and ammonia. Further, ifnecessary, inorganic salts such as calcium carbonate, sodium chloride,potassium chloride, and phosphates can be added; it is also possible tosuitably add organic or inorganic salts having an action to assist thegrowth of the microoragnism and encourage the production of the 5057Bsubstance. The accumulation of the 5057B substance produced reaches themaximum usually 4-8 days after the start of the cultivation in bothcases of shaking and tank cultures.

The 5057B substance is recovered from the culture broth by utilizing itsphysiochemical properties; since the substance is lipo-soluble andacidic, there can be used, in a suitable combination, extraction with avariety of organic solvents, chromatography on a variety of activatedadsorbents, and other methods. For example, an isolation methodconsisting of a combination of extraction and chromatography is carriedout as follows: The culture broth, after addition of a filter aid suchas diatomaceous earth and Radiolite 700 and the like, is filtered, andthe filtrate and the microbial cells are each extracted with suitablesolvents such as ethyl acetate and acetone. The cell extract and thefiltrate extract are combined. Evaporation of the solvent from thecombined extracts leaves, as the residue, crude crystals of a mixture ofthe 5057B and 5057A substances. The mixture is subjected to, forexample, chromatography to isolate the 5057B substance from the mixture.The eluate is concentrated under reduced pressure and then the residuetreated, in a suitable solvent, for example ethyl acetate, with a dilutehydrochloric acid solution and then with a dilute sodium carbonatesolution and so on to afford crystals of sodium salt of the 5057Bsubstance. Recrystallization of the crystals from a suitable solventsuch as n-hexane-ethyl acetate gives pure sodium salt of the 5057Bsubstance.

Properties of the sodium salt of the 5057B substance thus obtained areas follows:

(1) Colorless needles (the 5057B substance itself is acidic)

(2) Melting point: 143°-145° C.

(3) Elementary analysis (Found %): C, 59.96; H, 9.03; O, 27.60; Na,3.41.

(4) Specific rotation: [α]_(D) ²⁵ =+5.5° (C 1, CHCl₃)

(5) Ultraviolet absorption spectrum: The absorption spectrum taken in a0.25% methanol solution is shown in FIG. 1. Maximum absorption: λ_(max)^(MeOH) (E₁ cm^(1%)) 290 nm (0.88)

(6) Characteristic absorption (cm⁻¹) in infrared absorption spectrum(taken with the potassium bromide tablet): 3480, 3000, 2970, 2812, 1719,1645, 1595, 1465, 1385, 1160, 1100, 1035, 980 The infrared absorptionspectrum is shown in FIG. 2.

(7) Nuclear magnetic resonance spectrum (taken in CDCl₃) Proton NMRspectrum is shown in FIG. 3.

(8) Solubility: soluble in methanol, ethanol, ethyl acetate, chloroform,ether, acetone, and benzene and so on; insoluble in water

(9) Color reaction: positive in the 2,4-dinitrophenylhydrazine reaction;positive in the ninhydrin reaction and the vanillinesulfuric acidreaction; colors with I₂ gas

(10) Thin layer chromatography silica gel: Kieselgel GF₂₅₄ manufacturedby Merck Co.

    ______________________________________    Solvent system      Rf value    ______________________________________    chloroform-methanol (20:1)                        0.30    ethyl acetate-methanol (20:1)                        0.64    n-hexane-ethyl acetate (1:2)                        0.62    benzene-ethyl acetate (1:1)                        0.37    ______________________________________

(11) The antibacterial spectrum is shown in the following table:

    ______________________________________                          Minimum inhibitory                          concentration    Test microrganism       (mc.sup.g /ml)                                    Medium    ______________________________________    Staphylococcus aureus FDA 209 PJC-1                            0.5     a    Staphylococcus aureus    resistant to penicillins, strepto-                            0.5     a    mycin, kanamycin, chloramphenicol,    and tetracyclines    Bacillus subtilis ATCC-6636                            0.5     a    Sarcina lutea NIHJ      2       a    Micrococcus luteus ATCC-398                            0.5     a    Mycobacterium smegmatis ATCC-607                            5       a    Mycobacterium tuberculosis H.sub.37 R.sub.v                            10      b    Escherichia coli NIHJ JC-2                            100     a    Escherichia coli    resistant to streptomycin,                            100     a    kanamycin, chloramphenicol, and    tetracyclines    Klebsiella pneumoniae GN-6445                            100     a    Proteus vulgaris GN-75  100     a    Pseudomonas aeruginosa IFO-3756                            100     c    Penicillium citrinum ATCC-9849                            100     c    Aspergillus fumigatus NI-5561                            100     c    Alternaria kikuchiana CBS                            100     c    Ophiobolus miyabeanus ITO                            50      c    Nocardia asteroides     5       d    Candida albicans        50      d    ______________________________________

In the table, the sign a means the heart infusion agar medium, the signb the Kirchner's semifluid agar medium, the sign c the potato-sucroseagar medium, and the sign d the Sabouraud's 3% glucose agar medium. Ascan be seen from the antibacterial spectrum, the 5057B substanceexhibits a potent antibacterial activity especially againstgram-positive microorganisms.

(12) Acute toxicity test Acute toxicity tests were performed with mice.As a result, the LD₅₀ value was 50 mg/kg in the case of theintraperitoneal administration and 500 mg/kg or more in the case of theoral adminstration.

Melting points, specific rotations and Rf values on silica gel thinlayer chromatography of the 5057A and 5057B substances are shown in thefollowing table:

    ______________________________________             5057A substance                          5057B substance             (as the Na salt)                          (as the Na salt)    ______________________________________    Melting point               133-135° C.                              143-145° C.    Specific rotation               +1.0 (C 1, MeOH)                              +5.5 (C 1, CHCl.sub.3)    Rf value (1)               0.56           0.62    Rf value (2)               0.28           0.37    ______________________________________

The solvent systems used for the silica gel chromatography weren-hexane-ethyl acetate (1:2) and benzene-ethyl acetate (1:1) in (1) and(2), respectively.

The 5057B substance is useful as an antibacterial agent since it has anantibacterial activity.

THE BEST MODE OF CARRYING OUT THE INVENTION

The Streptomyces sp. 5057 strain (FERM-P No. 62) was inoculated into 1 lof a medium (pH 6.0) composed of soluble starch 6.0%, soybean meal 2.5%,beer yeast 0.5%, potassium primary phosphate 0.5%, ammonium sulfate0.3%, and calcium carbonate 0.3% and cultivation was carried out at 30°C. for 48 hours. The culture broth was inoculated into 100 ml of amedium having the same composition as set forth above and cultivationwas carried out with stirring under aeration in a 200-l tank at 30° C.for 96 hours. The rate of aeration was 100 l/min and the rotation rateof the 250 rpm.

The culture broth, after addition of a filter aid (Radiolite 700:registered trademark of Showa Chemical Industry Co.) was filtered toseperate the filtrate and cells. The filtrate was extracted with 40 l ofethyl acetate and the cells with 30 l of acetone. The acetone extract ofthe cells, concentrated under reduced pressure to remove the acetone,was extracted with 20 l of ethyl acetate. The extract thus obtained wascombined with the foregoing extract of the filtrate and the mixtureconcentrated under reduced pressure. The residue was adsorbed on acolumn in which 200 g of activated alumina had been packed with the useof ethyl acetate and a mixture of ethyl acetate-ethanol (1:1) passedthrough the column. Active fractions were concentrated to dryness underreduced pressure to give a mixture of the 5057B and 5057A substances inthe state of crude crystals. The mixture thus obtained was applied ontoa column in which 500 g of silica gel had been packed with the use ofn-hexane-ethyl acetate (1:2) and the same solvent passed through thecolumn. Fractions obtained were subjected to silica gel thin layerchromatography, which was developed with n-hexane-ethyl acetate (1:2).Only elute fractions containing the 5057B substance were collected andconcentrated under reduced pressure. The 5057A substance was elutedafter the 5057B substance had been eluted. The residue, dissolved inethyl acetate, was shaken together with dilute hydrochloric acid andthen the ethyl acetate layer shaken together with a dilute sodiumcarbonate solution. The ethyl acetate layer was then concentrated underreduced pressure. Crystals formed were recrystallized fromn-hexane-ethyl acetate to give 0.85 g colorless needles of sodium saltof the 5057B substance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows ultraviolet absorption spectrum of sodium salt of the 5057Bsubstance taken in a 0.25% methanol solution, FIG. 2 infrared absorptionspectrum of sodium salt of the 5057B substance, and FIG. 3 proton NMRspectrum of sodium salt of the 5057B substance.

What is claimed is:
 1. A new antibiotic 5057B which is itself an acidic substance or the sodium salt thereof which, as the sodium salt, has the following physiochemical properties:(1) Colorless needles (2) Melting point: 143°-145° C. (3) Elementary analysis (Found %): C, 59.96; H, 9.03; O, 27.60; Na, 3.41. (4) Specific rotation: [α]_(D) ²⁵ =+5.5° (C 1, CHCl₃) (5) Maximum absorption in ultraviolet absorption spectrum: λ_(max) ^(MeOH) (E_(1cm) ^(1%)) 290 nm (0.88) (6) Characteristic absorption (cm⁻¹) in infrared absorption spectrum (taken with the potassium bromide tablet: 3480, 3000, 2970, 2812, 1719, 1645, 1595, 1465, 1385, 1160, 1100, 1035, 980 (7) Solubility: soluble in methanol, ethanol, ethyl acetate, chloroform, ether, acetone, and benzene and so forth; insoluble in water (8) Color reaction: positive in the 2,4-dinitrophenylhydrazine reaction; negative in the ninhydrin reaction and the vanilline-sulfuric acid reaction; colors with I₂ gas.
 2. Antibiotic 5057B according to claim 1 in the form of the sodium salt. 